Despite improvements in early post-transplant survival rates over the last two decades, a relentless annual attrition rate of 3-5 % in recipients of previously successful renal allografts continues to limit longer term outcomes. Long term outcomes with islet transplantation are simply unacceptable with only 10% of recipients remaining insulin free at five years. In islets metabolic exhaustion due to an inadequate islet cell mass may be an important impediment to long term graft function. Late allograft failure, resulting from chronic rejection, infection, drug toxicity, and malignancies emphasizes the limitations of chronically administered immunosuppression in kidney and islet transplantations. Therefore the ultimate goal of transplantation is to achieve long-term engraftment without maintenance immunosuppression. Pilot clinical tolerance protocols are currently being tested in humans, but there remain substantial barriers to achieving true tolerance in humans. The major objective of this Multi-Project grant is to improve the outcome following kidney and islet transplantation by defining the essential conditions for induction of durable tolerance to kidney and islet allografts, and defining the roles of inflammation and memory T cells in being major barriers to tolerance. Our central hypothesis is that the early pro-inflammatory responses due in large measure to ischemia-reperfusion and anoxic injury to the donor tissues incites adverse forms of anti-donor immunity, thereby provoking acute clinical or subclinical rejection and exaggerating the subsequent expansion of pre-existing donor reactive memory, and post transplant development of newly acquired donor reactive memory T cells. Thus, we hypothesize that an adverse balance of pro- to anti-inflammatory cytokines and anti-donor memory responses represent major obstacles to the induction and maintenance of tolerance. We propose novel and inter-related strategies to alter the balance of the alloimmune response to favor regulation and long-term tolerance taking into consideration the inflammatory- and memory-related barriers to tolerance in the context of islet and kidney transplantation. The rationale linking the specific aims of the two interrelated in vivo projects and the mechanistic studies is that we now have the tools to test the relevance and inter-relationship of inflammatory responses to the balance of aggressive and memory responses, T cell regulatory and tolerance induction. It, therefore, should be possible to define and systematically apply the perturbations of the innate and adaptive immune response that lead to tolerance in primate allograft recipients. Moreover, the Program will lead to cross-fertilization and sharing of facets of the best tolerance inducing regimens developing from Project 1 with those of Project 2. PROJECT 1: Novel Tolerance Strategy in Renal Allograft Recipients (Cosimi, A. Benedict) PROJECT 1 DESCRIPTION (provided by applicant): Despite improvement in short-term results through the use of new immunosuppressive agents, long-term allograft survival has not improved significantly over the past decade, due predominantly to chronic rejection but also to infection and post-transplant malignancies, all attributable to chronic immunosuppression. Therefore, induction of specific transplantation tolerance, which might eliminate most limitations of conventional immunosuppressive therapy remains a major goal. Based upon the mixed chimerism approach, which was first demonstrated to be an effective means of inducing allograft tolerance in mice, we first developed a clinically relevant non-myeloablative preparative regimen that permitted the induction of mixed chimerism and renal allograft tolerance following donor bone marrow transplantation (DBMT) in MHC fully mismatched cynomolgus monkeys. More recently, this was applied clinically to recipients of HLA-identical and haplo-type identical kidneys. A disadvantage of the current preparative regimen is the requirement for conditioning beginning 6 days prior to organ transplantation, making it applicable only for recipients of living donor allografts and only prospectively with respect to the organ transplant. The major goal of this project is to develop a novel regimen, the "Delayed Tolerance" protocol to extend the clinical applicability of the mixed chimerism approach. In this protocol, recipients would initially undergo kidney transplantation (KTx) with conventional immunosuppression and then receive non-myeloablative conditioning and DBMT sometime later. This protocol could potentially extend the applicability to all recipients of previously transplanted allografts, including recipients of organs from deceased donors, if DBM is cryopreserved at the time of initial organ procurement from the deceased donor. This approach could prove to be even more effective than our current approach, since the tolerance conditioning regimen is instituted in the absence of pro-inflammatory cytokines that may impair tolerance induction in the peritransplant period. On the other hand, if tolerance induction is delayed, it might be predicted that the presence of the renal allograft could result in sensitization to donor antigen with memory T cell activation increasing. Therefore, to establish the "Delayed Tolerance" protocol, we will specifically: 1) identify the optimal timing for tolerance induction with minimal inflammatory responses, yet least likelihood of memory T cell activation, 2) evaluate methods to overcome memory T cell responses and 3) evaluate the addition of anti-inflammatory agents to the mixed chimerism protocol. Detailed mechanistic studies will be performed and the analyses planned should provide clinically relevant information for rationally developing new tolerance strategies for not only kidney but also all allograft recipients.